Osteoporosis, which has been defined as a “state of low bone mass” is one of the major aging problems of the society. Osteoporosis is a metabolic disorder characterized by microarchitectural deterioration of bone tissue leading to enhanced bone fragility and consequent increase in fracture risk in older members of the population. Osteoporosis fractures occur most commonly in the spine, hip, distal radius and ribs. The risk is high in women as compared to men and increases sharply after 50 years of age. Factors predisposing towards osteoporosis include family history, genetic factors, hormonal factors, inadequate nutrition, and intake of certain medications, immobility and disease. The quality of life is greatly impaired in persons with sever osteoporosis. It is known to affect >50% of women and 30% men over the age of 50 years. In women, there is also an accelerated rate of bone loss immediately and for variable number of years following menopause.
Most of the pharmacological agents available for clinical use such as calcium, vitamin D and its analogue, calcitonin, bisphosphonates, raloxifene, hormone replacement therapy (HRT) etc. act by decreasing the rate of bone resorption, thereby slowing the rate of bone loss. Timely administration of such antiresorptive agents prevents bone loss.
Hormone replacement therapy, though effective in preventing bone loss following ovariectomy or menopause in women, is associated with increased risk of endometrial hyperplasia and carcinoma [Grady, D. Grebretsadik, T. Ernestwr, V. Petitti, D. Gynecol. 85, 304-313 (1995), Beresford S. A. Weiss, N. S. Voigt, L. F. McKnight, B. Lancet 349, 458-461 (1997)], breast cancer [Riggs, L. Hartmann, L. C. J. Med. 348, 618-629, (2003)], and thromboembolic diseases [Delmas, P. D. Lancet 359, 2018-2026 (2002)].
The only side effect of calcium therapy is development of renal stones. The major disadvantage in calcitonin use is its high cost. Tachyphylaxis can develop in some individuals under calcitonin treatment. Bisphosphonates are poorly absorbed and may cause gastrointestinal irritation, diarrhea and constipation. Raloxifene has been reported to increase incidence of hot flashes, deep vein thrombosis, pulmonary embolism and leg cramps [Clemett, D.; Spencer, C. M. Drugs 60, 380-409 (2000)].
In view of the use of these therapies and their associated side effects indicate a need for the alternative options in the prevention and treatment of osteoporosis.
Traditional medicine is an ancient medical practice that existed in human societies before the application of modern science to health. The importance of traditional medicine as a source of primary health care was first officially recognized by the World Health Organization (WHO) in 1976 by globally addressing its Traditional Medicine Programme. In traditional medicine, there are many natural crude drugs that have the potential to treat bone diseases. However, not much laboratory work has been reported evaluating their possible development and use, except ipriflavone, a natural product derivative, which has been used clinically for such indications [Fujita, T.; Yoshikawa, S.; Ono, K.; Inoue, T.; Orimo, H. J. Clin. Exp. Med. 138, 113-141 (1986), Passeri, M.; Biondi, M.; Costi, D.; Bufalino, L.; Castiglione, g. N.; DiPeppe, C.; Abate, G. Bone Miner. 19 (Suppl. 1), S57-62 (1992)]. It is believed that herbal medicines are easily available, less expensive, and safer than chemically synthesized drugs. In India Ayurvedic medicine emerged during the rise of the philosophies of the Upanishads, Buddhism, and other schools of thought in India. Herbs played an important role in Ayurvedic medicine. In our program search for natural osteogenic plant, n-butanol soluble fraction of ethanol extract of Dalbergia sissoo aerial part which is renewable source exhibited osteogenic activity in our test model. Thus, the plant extract might possess bioactive ingredients that could promote bone formation. The effects on osteoporosis and total osteo-health and related disorders and has not been explored.
There is a well-recognized link between the prevalence of low peak bone mass (PBM) attainment and osteoporosis among South Asian women [Adami, S.; Osteoporos Int, Suppl 1, S27-30, (1994)]. PBM is defined as the highest level of bone mass achieved as a result of normal growth. Adolescence is the most critical period across the life span for bone health because more than half of PBM is accumulated during the teenage years. During these early years of life, bone formation is greater than bone resorption and the bone mass increases. PBM attained in early adult life is an important determinant of skeletal fragility at least until the age of 70 years (Ref). Following the attainment of PBM, resorption is faster than formation and the bone mass decreases. While gradual bone loss is normal to aging, it is those who fail to achieve optimal PBM and/or those with accelerated bone loss who are at the greatest risk of osteoporosis. In addition, low PBM predisposes to increased fragility fracture risk (Bonjour, J. P.; Chevalley, T.; Ferrari, S.; Rizzoli, R. Salud Publica Mex. 51 Suppl 1:S5-S17 (2009)].
Therefore, since individuals with a high PBM at a young age are likely to have a high bone mass in old age, agents increasing PBM during skeletal growth is a desirable goal towards prevention of osteoporosis. PBM occurs several years after the completion of linear growth as bone mineral accretion continues after this time, although the precise timing of the attainment of PBM is not certain and varies between skeletal sites. A real BMD at the femur peaks around the age of 20 yr, whereas maximum total skeletal mass occurs 6-10 yr later, well after the cessation of the anabolic action of growth hormone (GH). Factors relating to the attainment of PBM include congenital, dietary, hormonal, physical activity, lifestyle, drugs and diseases. A therapeutic intervention aimed at increasing PBM has remained limited only to controlling factors such as estrogen status, dietary calcium intake and physical activity. Calcium intake appears to be relevant up to the so-called threshold intake (1000 mg/day), but higher allowances do not seem to offer additive advantages. Exercise affects only the regions of the skeleton under mechanical stress. Estrogen administration is realistic only in conditions characterized by severe hypoestrogenism. Clearly, nutritional deficiency is one of the major reasons for lack of PBM among South Asians, particularly among females those who are much more prone to bone loss at later stages of life. Therefore, agents that promote PBM have therapeutic implication for bone loss disorders.
There is, thus, an urgent need to discover and develop a promising herbal product or a single biologically active molecule based drug or a cocktail of the pure and biologically active molecules of the plant origin that exhibit promising bone anabolic or for bone forming activity in experimental animals and human beings. The Dalbergia sissoo was a fit case to study and explore its true potential with respect to its bone forming response of its extract, fraction and pure biologically active marker components. The experiments have shown that its n-butanol soluble fraction and pure compounds isolated from the extract and the fraction exhibit promising bone forming activity.
Dalbergia sissoo Roxb. belongs to the family Fabaceae, is distributed throughout sub-Himalayan tract from Ravi to Assam ascending up to 5000 ft in India, Pakistan, Bangladesh and Afghanistan. Dalbergia sissoo, commonly known as “Shisham” in India, is deciduous tree, having crooked trunk and light crown [Wealth of India. Raw materials, vol 3. CSIR, New Delhi, 1950.] An aqueous extract of the leaves of Dalbergia sissoo has been used for the treatment of gonorrhoea [Medicinal plants of India; S. K. Jain, Roberts A. Defilipps; 1991. vol-1, 325]. Leaves of the plant are bitter and stimulant and also used as fodder. Wood of Dalbergia sissoo was used for leprosy [R. N. Chopra, S. L. Nayar, I. C. Chopra. Glossary of Indian Medicinal Plants, 1956, page 90].
The methylene chloride extract of the heart wood of Dalbergia sissoo inhibited the production of β-amyloid peptides (Aβ) so it may have therapeutic potential in the treatment of Alzheimer's disease. A compound latifolin, isolated from the crude extract of the heart wood also inhibits β-amyloid production [Ramakrishna, N. V. S., Kumar Vijaya, E. K. S., Kulkarni, A. S., Jain, A. K., Bhat, R. G., Parikh, S., Quadros, A., deuskar, N., Kalakoti, B. S., Indian Journal of Chemistry 40B, 539-540, 2001]. Heartwood of the plant has also been reported to have anthelmintic activity [Manandhar, N. P., Fitoterapia 2, 149, 1995].
The ethanolic extract of the leaves of Dalbergia sissoo has anti-inflammatory activity [Hajare, S. W., Chandra, S., Sharma, J., Tandan, S. K., Lal, J., Telang, A. G., Fitoterapia 72 (2), 131-139, 2001] and petroleum ether extract also display the same activity due to a sterol i.e. sissosterol [Abdel-Ghani, Afaf, E., Dora, Gamal A., Mansoura journal of pharmaceutical science, 20(1), 104-113, 2004]. It has also been reported to possess antidiabetic property in alloxan-induced diabatic rats [Niranjan, P. K., Singh, D., Prajapati, K., Jain, S. K., International Journal of Current Pharmaceutical Science 2(2), 24-27, 2010]. The leaves of Dalbergia sissoo exhibit antipyretic, analgesic properties [Hajare, S. W., Chandra, S., Tandan, S. K., Sharma, J., Lal, J., International Journal of Pharmacology 32, 357-360, 2000] and also acts against diarrhea [Brijesh, S., Deswani, P. G., Tetall, P., Antia, N. H., Birdl, T. J., Indian J. Pharmocol. 38(2), 120-124, 2006]. The preparation of Dalbergia sissoo leaves has been used as an alternative herbal treatment for antimicrobial property [Yadav, H., Yadav, M., Jain, S., Bhardwaj, A., Shing, V., Parkash, O., Marotta, F., International Journal of Immunopathology and Pharmocology 21 (4), 1013-1020, 2008]. An methanolic extract from the roots of Dalbergia sissoo has been reported to have anti-inflammatory activity in carrageenan-induced paw edema in rats [Kumar, S. M., Kumud, U., Pharmacognosy journal 2(11), 427-430, 2010]. The alcoholic extract of green branches of aerial showed a dose dependent inhibitory effect on the motility of isolated rabbit duodenum or bronchodilation and significant anti-inflammatory, antipyretic, analgesic, estrogenic activities [Sarg, T., Ateya, Abdel-Monem, Abdel-Ghani, A., Badr, W., Shams, G., Pharmaceutical Biology 37(1), 54-62, 1999].
The alcoholic and chloroform extract of the bark of Dalbergia sissoo have been reported for anti-inflammatory, anti-ulcerogenic and antioxidant activities and the compound dalbergin isolated from the bark is found to possess same activities [Khaleel, A. E., El-Gayed, S. H., Ameen, A., Al-Azhar Journal of Pharmaceutical Sciences 28, 285-299, 2001]. The bark has been also tested for antioxidant potential in an in vitro assay [Kumari, A., Kakkar, P., Biomedical and Environmental sciences 21(1), 24-29, 2008].
The anti-inflammatory, anti-ulcerogenic and antioxidant activities have been reported in alcoholic and chloroform extract of flowers of Dalbergia sissoo and the 7-methyltectorigenin, was found to have active constituent for these activities [Khaleel, A. E., El-Gayed, S. H., Ameen, A., Al-Azhar Journal of Pharmaceutical Sciences 28, 285-299, 2001].
Pure Compounds
A variety of compounds have been isolated from different parts of Dalbergia sissoo. The mature pod of Dalbergia sissoo contains isocaviunin and 7-hydroxy-4-methyl coumarin [Sharma, A., Chibber, S. S., Chawla, H. M., Indian Journal of Chemistry 18B, 472-473, 1979]. 4′-Rhamnoglucoside of 7-methyltectorigenin and meso-inasitol has been isolated from immature pods of Dalbergia sissoo [Ahluwalia, V. K., Sachdev, G. P., Seshadri, T. R., Indian Journal of Chemistry 3, 474, 1965]. A new isoflavane glucoside, isocaviudin among with tectoridin and caviunin 7-O-β-glucoside has been found as constituent of immature pods of Dalbergia sissoo [Sharma, A., Chibber, S. S., Chawla, H. M., Indian Journal of Chemistry 19B, 237-238, 1980]. Two isoflavone glycosides caviunin 7-O-gentiobioside and isocaviunin 7-O-gentiobioside has been isolated from the mature pods of Dalbergia sissoo. [Sharma, A., Chibber, S. S., Chawla, H. M., Phytochemistry 18, 1253, 1979; Sharma, A., Chibber, S. S., Chawla, H. M., Phytochemistry 19, 715, 1980].
From the flowers of Dalbergia sissoo, isoflavones, 7-methyltectorigenin along with tectorigenin, prunetin, kaempferol have been isolated and compound 7-methyltectorigenin has shown anti-inflammatory, anti-ulcerogenic and antioxidant activities [Banerji, A., Murti, V. V. S., Seshadri, T. R., Indian Journal of Chemistry 1, 25-27, 1963; Khaleel, A. E., El-Gayed, S. H., Ameen, A., Al-Azhar Journal of Pharmaceutical Science 28, 285-299, 2001]. Biochenin A, a potent cancer preventive agent with estrogenic activity has been isolated in good yield from the fresh flower of Dalbergia sissoo [Asaab, Aya M., El-Shaer, Nagwa, S., Darwish, F., Alexandria Journal of Pharmaceutical Science 14(2), 103-105, 2000]. 7,4′-Dimethyltectorigenin has also been isolated from the flowers of Dalbergia sissoo [Banerji, A., Murti, V. V. S., Seshadri, T. R., Current Science 34(14), 431, 1966].
A chalcone 2,3-dimethoxy-4′-γ,γ-dimethylallyloxy-2′-hydroxychalcone, two isoflavone 7-γ,γ-dimethylallyloxy-5-hydroxy-4′ methoxyisoflavone and biochenin, a flavone, 7-hydroxy-6-methoxyflavone and a rotenoid have been isolated from the root bark of Dalbergia sissoo [Reddy, R. V. N., Reddy, N. P., Khalivulla, S. I., Reddy, M. V. B., Gunasekar, D., Blond, A., Bodo, B., Phytochemistry Letters 1(1), 23-26, 2008]. The chloroform extract of bark of Dalbergia sissoo contain dalbergin while ethyle acetate extract shown the presence of tectorigenin, tectorigenin-7-O-apioglucoside, tectorigenin-4′-O-apioglucoside, stearic acid and palmitic acid [Ragab, A., Mostafa, S. M. I., El-Shami, I., Ibrahim, Abdel-Rahim S., Mansoura Journal of Pharmaceutical Science 22(2), 176-194, 2006]. Two aliphatic esters, n-hexacosan-5-ol-1-yl-propionate, n-teracosan-5-ol-yl-propionate and two pyran 7-hydroxy-8-methoxy-4-(2′-hydroxyphenyl)[4H]benzopyran, 10,12,13,trihydroxy-11-methoxyanthracenyl-15-18[2H]pyran have been isolated from ethanolic extract of stem bark of Dalbergia sissoo Roxb [Trag, A. R., Ali, M., Siddiqui, T. O., Mahmooduzzafar, Iqbal, M., Journal of Saudi Chemical Society 9(2), 341-345, 2005]. Dalbergin has significant anti-inflammatory activity, anti-ulcerogenic antioxidant activity [Khaleel, A. E., El-Gayed, S. H., Ameen, A., Al-Azhar Journal of Pharmaceutical Science 28, 285-299, 2001]. 4-Arylcoumarin and fisetin have also been isolated from the bark [Khaleel, A. E., El-Gayed, S. H., Ameen, A., Al-Azhar Journal of Pharmaceutical Science 28, 285-299, 2001]. Isotectorigenin was isolated from the bark of Dalbergia sissoo [Dhingra, V. K., Seshadri, T. R., Mukerjee, S. K., Indian Journal of Chemistry 12(10), 1118, 1974]. Dalbergenone, methyldalbergin, dalbergichromene were also known constituent of bark of Dalbergia sissoo. 
Irisolidone, biochenin-A, muningin, tectirigenin, prunetin, genistein, sissotrin, prunetin-4-O-galactoside, norartocarpetin, β-amyrin, β-sitosterol, and stigmasterol along with 13 fatty acids were isolated from the green branches of aerial parts of Dalbergia sissoo Roxb [Sarg, T., Ateya, Abdel-Monem, Abdel-Ghani, A., Badr, W., Shams, G., Pharmaceutical biology, 37(1), 54-62, 1999].
Biochenin A, kaempferol, quercetin, kamempferol-3-O-β-D-glucoside, quercetin-3-α-L-rhamnoside, rutin, β-sitosterol were isolated from the leaves of Dalbergia Sissoo [Ragab, A., Mostafa, S. M. I., El-Shami, I., Ibrahim, Abdel-Rahim S., Mansoura journal of Pharmaceutical Science 22(2), 176-194, 2006].
The Oligosaccharides were also isolated from the leaves of Dalbergia sissoo [Rana, V., Kumar, V., Soli, P. L., Carbohydrate polymers 78(3), 520-525, 2009]. Sissosterol isolated from petroleum ether extract of Dalbergia sissoo leaves has also been reported to show significant antiinflmmatory activity [Abdel-Ghani, Afaf, E., Dora, Gamal A., Mansoura journal of pharmaceutical science, 20(1), 104-113, 2004]. Biochenin A 7-O-[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside, Biochenin A 7-O-[β-D-apiofuranosyl-(1→5)-β-D-apiofuranosyl (1→6)-β-D-glucopyranoside], tectorigenin 7-O-[β-D-apiofuranosyl(1→6)-β-D-glucopyranoside], prunetin 4′-O-[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside], 7-methyltectorigenin 4′-O-[β-D-apiofuranosyl-(1→6)-β-D-glucopyranoside], genistein 8-C-[β-D-glucopyranoside] and prunetin 4′-O-[β-D-glucopyranoside] have also been isolated from the leaves of Dalbergia sissoo [Farag, S. F., Ahmed, A. S., Terashima, K., Takaya, Y., Niwa, M., Phytochemistry 57(8), 1263-1268, 2001]. Sissotrin has been reported from the leaves [Banerji, A., Murti, V. V. S., Seshadri, T. R., Indian Journal of Chemistry, 4(2), 70-72, 1966].
Trunk exudates of Dalbergia sissoo contain S-4′-hydroxy-4-methoxy-dalbergione, S-4-methoxydelbergone, S-dalbergion, S-4-methoxydelbergiqninol, 4-[(1S)-1-phenyl-2-propenyl]-1,3-benzenediol, (4S,6S)-4-hydroxy-3-methoxy-6-(1-phenyl-2-propenyl)-2-cyclohaxene-1-one, (4S,6S)-4-hydroxy-6-(1-phenyl-2-propenyl)-2-cyclohaxene-1-one, (2S,4R,5S)-4-hydroxy-5-methoxy-2-[(1S)-1-phenyl-2-propenyl)]cyclohaxanone, (2S,4R,6S)-4-hydroxy-2-methoxy-6-(1-phenyl-2-propenyl)cyclohaxanone, (1S,2S,4S,5S)-2-methoxy-5-[(1R)(1-phenyl-2-propenyl)-1,4-cyclohaxanediol, cearoin, 4-hydroxy-3-methoxy-4-(3-phenyl-2-propenyl)-2-cyclohaxen-1-one, isoliquiritigenin, butein, 2′-hydroxy-4′-methoxychalcone, hydroxyobtustyrene, (2S)-7-hydroxyflavanone, (+)-pinocembrin, plathymenin, (±)-vestitol, dihydrosepiol, formononetin, zenognosin B, 4-hydroxymedicarpin, (+)-medicarpin, (+)-vesticarpan [Shrestha, S. P., Amano, Y., Narukawa, Y., Takeda, T., Journal of Natural Product, 71, 98-101, 2008].